DE102004004924B4 - A141S and G399S mutation in the Omi / HtrA2 protein in Parkinson's disease - Google Patents

Abstract

Nucleic acid molecule coding for a human Omi / HtrA2 protein, the at amino acid position 141 and / or 399 one opposite the wild-type genetic change has, as well as for corresponding sections thereof, for use in diagnosis Parkinson's disease and / or predisposition thereto and / or for finding and testing substances that are active against Parkinson's disease.

Description

The The present invention relates to a method for the diagnosis of morbus Parkinson in a human being; in this procedure used nucleic acid molecules as well their uses for the detection of a nucleic acid molecule encoding human Omi / HtrA2 protein or to amplify the human Omi / HtrA2 gene; the use of a nucleic acid molecule, the for a across from the wild type genetically modified Omi / HtrA2 protein or portions thereof, and / or such Proteins or sections thereof for the diagnosis of Parkinson's disease and / or an investment therefor; a nucleic acid molecule that is responsible for a human Omi / HtrA2 protein encoded at amino acid position 141 and / or 399 one opposite the wild-type genetic change has, as well as for corresponding sections thereof; a host, namely a transgenic non-human Mammal, into which such a nucleic acid molecule introduced has been; a (poly) peptide encoded by such a nucleic acid molecule; a method for finding genetically modified from the wild type Omi / HtrA2 protein binding substances; one by means of this method substance found and a preferably pharmaceutical composition, which has such a substance. Moreover, this invention relates to a Kit comprising at least one of the aforementioned nucleic acid molecules.

method of the type mentioned above are common in the art known.

at Parkinson's Disease or Parkinson's disease is the second most common neurodegenerative Disease in humans and the second most common neurological disease of people of advanced age. For those over eighty years old 4% affected, in Germany alone there are about 250,000 Parkinson's patients.

Cardinal symptoms Parkinson's disease is tremor, rigor and akinesia, i. Tremble, the maximum pronounced at rest is, slowing down and poverty, difficulty in the Initiation of movements, small-step gait in front over bent Upper body, extinguished postural reflexes with falling tendency, mask-like facial expression, quiet, monotonous and stuttering language. In about 50% of patients developed a dementia.

The Parkinson's disease also goes with a degeneration of dopamine-forming cells in the substantia nigra pars compacta, presumably via a apoptotic cell death, resulting in functional dysbalance downstream cores leads. Because dopamine is the activity of nerve cells in different brain areas, the leads Dopamine loss to overstimulation of these brain regions.

The etiology Parkinson's Disease is largely unknown. In doing so, environmental factors, such as pesticides, as influence on the onset of this disease discussing factors.

on the other hand genetic factors play a significant role in a disease at Parkinson's Illness. These findings are based on twin studies (cf. see Piccini et al. (1999), The role of inheritance in sporadic Parkinson's disease: evidence from a longitudinal study of dopaminergic function in twins. Ann. Neurol., 45, 577-582), the identification of big ones from Parkinson's Disease affected families (see, for example, Nicholl et al. (2002), Two large British children with familial Parkinson's disease: a clinico-pathological and genetic study. Brain, 125, 44-57), and increased risk in relatives of so-called index patients to Parkinson's disease too (see Elbaz et al. (1999), Familial aggregation of Parkinson's disease: a population-based case-control study in Europe. EUROPARKINSON Study Group. Neurology, 52, 1876-1882).

So far Ten gene loci identified with Parkinson 's disease were identified Connection stand. An overview here over can be found in Kruger et al. (2002), Parkinson's disease: one biochemical pathway to fit al1 genes ?, TRENDS in Molecular Medecine, Vol. 5, 236-240. Furthermore, four genes could be identified, α-synuclein, synphilin-1, Parkin and UCH-L1, which are inherited in autosomal dominant or autosomal recessive Parkinson's disease in mutated form. All gene products of this Genes are members of the proteasome protein degeneration pathway. While mutations in the α-synuclein, synphilin-1, and extremely rare in the UCH-L1 genes are found in nearly half of cases of autosomal recessive Forms of the so-called early-onset Parkinson's disease (AR-EOPD) Mutations in the Parkin gene; see. Lucking et al. (2000), Association between Parkinson's early-onset disease and mutations in the parkin genes. French Parkinson's Disease Genetics Study Group. N.Engl.J.Med., 342, 1560-1567.

In front Recently, two gene loci correlating with AR-EOPD were able to PARK6 and PARK7, the human chromosome lp35-36. During the underlying gene defect of PARK6 has not yet been identified Bonifati et al. the identification of a homozygous L166P substitution and a deletion in two not related families in the DJ-1 gene as a trigger of PARK7-associated Parkinson's Illness; see. Bonifati et al. (2003), mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science, 299, 256-259.

In the previously unpublished German patent application with the file number 103 48 904.5 about that another new AR-EOPD-related mutation described in the DJ-1 gene, by the one in the DJ-1 protein at amino acid position 64 a Glutaminsäuremolekül against exchanged an aspartic acid molecule becomes.

The Diagnosis of Parkinson's Disease or AR-EOPD so far mainly on the basis of the observed cardinal symptoms asked, what with corresponding uncertainties and risks of Misdiagnosed. Such a diagnosis can only be post-mortem by the neuropathological proof of the characteristic so called Lewy bodies, which are abnormal protein deposits in the brain, be secured. A predisposition for a corresponding illness Therefore, so far can not or only with a very large uncertainty factor be diagnosed. In the prior art therefore lacks the basis for one appropriately targeted preventive Treatment of affected persons, both medicinal and possibly psychotherapeutic.

task It is therefore an object of the present invention to provide a method with the above-mentioned disadvantages of the prior art be avoided. In particular, such a method should be provided be made by means of the affected persons a molecular biological Differential diagnosis of a disease or predisposition to morbus Parkinson possible is that, for example, can be embedded in the additional Proof of genetic changes, those in the prior art already correlate with Parkinson's disease have been described.

These Task is solved by providing a method for diagnosing morbidity Parkinson's in a human being, the following steps comprising: (a) providing a biological sample of the animal; (b) Examination of the biological sample for the presence of a Nucleic acid molecule and / or a (poly) peptide, and (c) correlation of a positive finding with a disease of Parkinson's disease and / or with a predisposition for one Disease of Parkinson's disease, wherein in step (b) the biological Sample is examined for the presence of such a nucleic acid molecule, that for encodes a human Omi / HtrA2 protein at amino acid position 141 and / or 399 one opposite the wild-type genetic change has, as well as for corresponding sections thereof.

The The object underlying the invention is hereby completely solved.

The Inventors could surprisingly show for the first time that a genetic change in the Omi / HtrA2 protein, preferably one at amino acid position 141 or 399, with Parkinson's disease correlates. This realization is all the more surprising since the Omi / HtrA2 gene or the protein encoded thereby not yet was associated with Parkinson's disease. The Omi / HtrA2 protein is it is a serine protease, which was first described in 2000; see. Faccio et al. (2000) Characterization of a novel human serine protease that has extensive homology to bacterial heat shock endoprotease HtrA and is regulated by kidney ischemia, The Journal of Biological Chemistry, Vol. 275 No. 4, pages 2581-2588; Gray et al. (2000), Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response, European Journal of Biochemistry Vol. 267, pp. 5699-5710. The sequence of the human Omi / HtrA2 gene can be found in the NCBI database: NM 013247.3 or NM 145074.1 (mRNA, respectively); AC005041.2 (genomic sequence, BAC); AF141305.1 or AF020760.2 (each cDNA).

The Omi / HtrA2 protein is a member of the HtrA protease chaperone family and located in the intermembrane space of the mitochondria. Omi / HtrA2 has been implicated in stress-induced cell death (apoptosis), and it has been shown that the serine protease activity of the Omi / HtrA2 protein is responsible for the mediation of caspase-independent cell death; see. Cilenti et al. (2003), Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi / HtrA2, Journal of Biological Chemistry, Vol. 278, pages 11489-11494. In addition, the Omi / HtrA2 protein is about interacting with the ubiquitin E3 league se XIAP closely linked to the ubiquitin-mediated protein degradation pathway; see. for example, Suzuki et al. (2001), A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death, Molecular Cell, Vol. 8, pp. 613-621.

Jones et al. (2003), Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice, Nature, Vol. 425, Pages 721-727, describe the first mutation found in the Omi / HtrA2 gene by those in the protein at amino acid position 276 a serine molecule against a cysteine molecule is exchanged. The mutation described there was in a identified with mnd2 and known since 1990 mouse mutant, the neuromuscular disorders and mitochondrial defects. The local authors were able to Try embryonic mouse fibroblasts mutant appropriately Omi / HtrA2 protein expressed an increased susceptibility of these cells to stress show induced apoptosis. A possible meaning of the Omi / HtrA2 protein or the genetic modification described there in Parkinson's disease is not recognized by the authors there. Even more, the local ones Authors have two others affected by Parkinson's disease in further studies Examined families for a corresponding mutation in the Omi / HtrA2 gene, could not find such a one; see. Jones et al. supra; especially page 726, left column, second paragraph.

at the doctrine recognized by the inventors is therefore a departure from the hitherto accepted view in the prior art, in the one connection of the Omi / HtrA2 gene with the clinical picture Parkinson's not only not recognized, but even denied becomes.

The On the other hand, inventors were able to obtain a Parkinson's disease in 29 patients with Parkinson's disease genetic change in the Omi / HtrA2 gene, but not in over 600 healthy controls. In parallel, the inventors made use of established toxicity tests the pathophysiological relevance of the found genetic changes recognized.

Of the Detection of a nucleic acid molecule in one human sample that for a genetically modified human Omi / HtrA2 protein encodes, thus according to the invention the diagnosis of a predisposition to or an already existing disease of Parkinson's disease. Even more so, after the first time the importance of the Omi / HtrA2 gene for a disease has been detected on Mobus Parkinson's can now targeted for more pathologically relevant genetic changes in this gene screened become.

A those to be investigated according to the invention biological sample can be any biological material that representative nucleic acids and / or proteins of the corresponding human being, eg. a blood, tissue, saliva, hair or other sample.

The Examination of the biological sample in step (b) takes place by means of in the state of the art well-known methods, for example by means of a mutation screening using e.g. PCR-based Methods and heteroduplex analyzes such as the denaturing high pressure liquid chromatography (dHPLC) or hybridization techniques apply.

As The inventors have recognized sufficient for a successful implementation of the inventive method the examination of the biological sample for the presence of Nucleic acid molecules, the for encode such Omi / HtrA2 protein sections that are the genetic change and one in total protein at positions 141 and 399, respectively corresponding amino acid residue exhibit. How the inventors were able to find out is just that a genetic change at this position in the Omi / HtrA2 total protein for the onset of Morbus Parkinson's vital.

In the method according to the invention encodes the nucleic acid molecule to be detected for a human Omi / HtrA2 protein, that at the amino acid position 141 or 399 an amino acid exchange, preferably has one, by which an alanine molecule against a serine molecule or a glycine molecule against a serine molecule is exchanged.

The investigation according to the invention of the biological sample for the presence of such a nucleic acid molecule has the advantage that a very significant and meaningful genetic modification or mutation is detected hereby for the correlation with Parkinson's disease. Surprisingly, the inventors were able to show in this connection that 29 patients suffering from Parkinson's disease carry at least one of these amino acid exchange mutations, whereas healthy control persons had no genetic alterations at position 399 or significantly less frequently at position 141. These found genetic changes are due to nucleotide or base changes to the posi tions 421 and 1195 of the open reading frame (ORF) of the human Omi / HtrA2 gene, in which compared to the wild type a deoxyguanosine monophosphate (dGMP) or guanine (G) against deoxythymidine monophosphate (dTMP) or thymine (T) or a dGMP or G was exchanged for a deoxyadenosine monophosphate (dAMP) or adenine (A).

Of the Detection of such a nucleic acid molecule as defined above by means of the method according to the invention allows therefore the reliable one Diagnosis of a predisposition for or a disease of Parkinson's disease.

The inventive method is purposeful even then if the nucleic acid molecule to be detected is such is that under stringent conditions to the previously described Nucleic acid molecule binds. Under stringent conditions, those known to those skilled in the art are known Understand conditions under which only perfectly base-paired Nucleic acid strands formed become and remain stable.

These measure has the advantage that, for example, the complementary non-coding strand of the human Omi / HtrA2 gene for the diagnosis of Parkinson's disease is used, whereby the sensitivity of the method is additionally increased.

In In this context, it is also possible in the inventive method, as to be detected nucleic acid molecule such which, under stringent conditions, in turn belongs to the here above mentioned nucleic acid molecule binds.

Of the Advantage of this measure is that even such nucleic acid molecules used for detection which derive from the ORF of the Omi / HtrA2 gene, such as. mature or immature mRNA, as well as degradation products thereof, however still carry the characteristic genetic change (s) and therefore bind to the nucleic acid molecule, that to the for the changed Omi / HtrA2 gene-encoding nucleic acid molecule is complementary. By This measure becomes the sensitivity the method according to the invention further increased.

In front This background is also the subject of the present invention encoding a nucleic acid molecule for a human Omi / HtrA2 protein at amino acid position 141 and / or 399 one opposite the Wild-type genetic modification, preferably an amino acid exchange, further preferably has such, by the position 141 an alanine molecule against a serine molecule and / or at position 399 a glycine molecule is replaced by a serine molecule is, as well as such nucleic acid molecules, the for corresponding Encode sections thereof. The nucleic acid molecule of the invention is for use in the Diagnosis of Parkinson's disease and / or predisposition and / or for finding and testing Parkinson's disease Provided substances.

On Reason of the first recognized by the inventors of a connection genetic change in the Omi / HtrA2 gene is also the subject of the present invention the use of the above-described nucleic acid molecules for diagnosis Parkinson's disease and / or predisposition thereto.

The The present invention further relates to a host, namely a transgenic non-human mammals, preferably a transgenic mouse, a transgenic rat, a transgenic Sheep, a transgenic goat or a transgenic cow, in the least one of the above-described nucleic acid molecules which is genetically modified for a wild-type Omi / HtrA2 protein encoded was introduced.

The key role described by different working groups of the Omi / HtrA2 protein in caspase-independent apoptosis and in the Ubiquitin dependent Protein degradation and the first recognized by the inventors Connection between Omi / HtrA2 and Parkinson's disease speak for a key role of this gene or of the protein encoded thereby in the molecular Pathogenesis in Parkinson's disease. This is also the case with Parkinson's disease observed neurodegenerative processes, which are usually not the classic Criteria of caspase mediated apoptotic or necrotic Correspond to cell deaths. Furthermore, in Parkinson's disease also a disorder described the protein degradation, which in the formation so-called. Lewy bodies in the brain of affected patients shows. The mechanisms responsible for these phenomena were so far unknown despite intensive research. By finding of disease-causing mutations in the Omi / HtrA2 gene in Morbus Parkinson's by the inventors are the first known pathogenesis pathways the disturbed Protein degradation associated with apoptotic processes. The inventors Furthermore, in further experiments a ubiquitination of the Omi / HtrA2 protein and the latter in characteristic Lewy bodies in the brain of affected patients.

through of the transgenic invention mammal Therefore, a model system is provided, with the molecular Pathology of Parkinson's disease more comprehensive than in the prior art can be replicated and analyzed. Of course such a transgenic host is also an excellent system for finding and testing of Parkinson's disease active substances. The for the production of a transgenic mammal, such as an Omi / HtrA2 knock-out mouse, Required process steps are included in the prior art described; see. for example, Thomas Rülicke (2001) Transgene, Transgenesis, transgenic animals: methods of non-homologous DNA recombination, Karger Publishers.

The Inventors have about it In addition, it is recognized that after a modification of the above-described new procedure also makes a diagnosis of Parkinson's disease possible, if indeed in the biological sample a (poly) peptide molecule is detected which is from one opposite genetically modified wild type Omi / HtrA2 protein is derived. This is the biological sample on investigated the presence of a (poly) peptidyl molecule, that of one of the nucleic acid molecules described above is encoded. As explained genetically changed Omi / HtrA2 protein is the translation product of the corresponding genetic changed Nucleic acid molecule and therefore leaves also direct conclusions an investment for or disease of Parkinson's disease.

at a variant of the method according to the invention Therefore, in step (b) the investigation of biological Assay for the presence of such a (poly) peptide derived from one of the nucleic acid molecules described above is encoded.

These measure has the advantage that upon detection of such a (poly) peptide an even stronger diagnosis of Parkinson's disease becomes possible for example, also possible if that is for the peptide-encoding nucleic acid molecule in the biological sample, e.g. due to nuclease activities, not or no longer detectable.

Of the Detection of the (poly) peptide with the genetic modification at the amino acid position 141 and 399, in the biological sample takes place by means of protein purification and / or optionally known in the art Sequencing. An overview of such Methods can be found, for example, in Lottspeich F. (publisher) et al. (1998), "Bioanalytics", Spectrum Academic Publishing company.

In front This background is also the subject of the present invention such a (poly) peptide as that of any of the previously described Nucleic acid molecules encoded becomes.

It It is understood that with such a (poly) peptide also such peptides are captured, the fragments, variants and isoforms of the genetic changed Represent Omi / HtrA2 protein, and those in the total protein in position 141 and 399 existing above explained genetic change exhibit.

One such (poly) peptide provides the basis for the development of pharmacological active substances, such as inhibitors or activators of the according to genetically modified Omi / HtrA2 protein. The same applies to those for such a poly (peptide) encoding nucleic acids via the eg pharmacologically interesting inhibiting RNAi (RNA interference) or siRNA (silencing RNA) molecules or other antisense molecules can be constructed. By means of the coding nucleic acid molecules can For example, the genetically modified Omi / HtrA2 protein in big Quantities produced, the three-dimensional structure cleared up and above means In silico-screenings pharmacologically active substances are developed.

at the method according to the invention described above, it is preferred if, in step (b), the investigation is for the presence of the person of interest Nucleic acid molecule by means of PCR technology is done.

These measure has the advantage of being an extremely sensitive, well-established and largely automatable method finds application over the highly specific the genetically modified nucleic acid material can be enriched, which then subsequently by means of other standard methods such as electrophoresis / heteroduplex or direct sequencing can be detected.

When PCR primer is preferably a nucleic acid molecule is used, which is one of the sequences which has selected is selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 17.

The inventors have surprisingly found that by means of the use of the above be recorded nucleic acid molecule with one of the listed nucleotide sequences a highly specifi cal and selective amplification of the entire Omi / HtrA2 gene succeeds and therefore can be screened for genetic changes in the entire open reading frame. As has been shown, with a PCR primer having the nucleotide sequence of SEQ ID NO: 1 as the forward primer and with a PCR primer having the nucleotide sequence of SEQ ID NO: 2 as the reverse primer, the amplification of exon 1 of the human Omi / HtrA2 gene. Accordingly, with PCR primers having the sequences SEQ ID No. 3 and SEQ ID No. 2 it is likewise possible to amplify the exon 1, with the sequences SEQ ID No. 4 and SEQ ID No. 5 the amplification of the exon 2, with the sequences SEQ ID No. 6 and SEQ ID No. 7 describe the amplification of exon 3, with the sequences SEQ ID No. 8 and SEQ ID No. 9 the amplification of exon 4, having the sequences SEQ ID No. 10 and SEQ ID No. 1. 11 the amplification of the exon 5, with the sequences SEQ ID No. 12 and SEQ ID No. 13 the amplification of the exon 6, with the sequences SEQ ID No. 14 and SEQ ID No. 15 the amplification of the exon 7, and with the Sequences SEQ ID NO: 16 and SEQ ID NO: 17 the amplification of the exon 8 of the Omi / HtrA2 gene.

At Hand of the amplification products can then any existing genetic change by means of usual Mutation screening method or hybridization method easy be detected.

Of course, succeed Such amplification also with such PCR primers, in addition to one of the sequences SEQ ID No. 1 to SEQ ID No. 17 is 5'- and / or 3'-further Have nucleotides which optionally hybridize with the opposite strand, or which have smaller sequence variations but which have the specificity the primer does not change significantly.

In front this background succeeds the corresponding inventive use even if a nucleic acid molecule as a PCR primer is used which binds under stringent conditions to a nucleic acid molecule, having one of the sequences selected from the group consisting from: SEQ ID No. 1 to SEQ ID No. 17.

Also a stringent condition to a nucleic acid molecule having a The sequence of SEQ ID NO: 1 to SEQ ID NO: 17 binding nucleic acid molecule allows the highly specific and selective amplification of a section of the Omi / HtrA2 if necessary. genetically changed Omi / HtrA2 gene, but here hybridization with the corresponding complementary Strand occurs, but the amplicons are largely identical.

On Reason for the unexpected characteristics and particular suitability of the The novel nucleic acid molecules described above are the subject of the present invention Invention also their use for amplifying the human Omi / HtrA2 gene.

at the above-explained inventive method it is preferred that the PCR amplicons are denaturing by means of denaturing High pressure liquid chromatography (dHPLC) or other heteroduplex method.

in this connection it is a procedure that uses high sensitivity sequence variations in PCR amplicons compared to the wild-type sequence can. The detection is based on a heteroduplex that differs artificial de-naturing and renaturation always forms, if beside the wild type Omi / HtrA2 allele the sequence variation in question present on the second allele. Therefore, to prove homozygous Omi / HtrA2 mutation before artificial denaturation by heating and slow Cooling additionally "wild-type material" added The previous PCR is produced with high reliability a heteroduplex from one strand of the wild-type Omi / HtrA2 allele and one strand of the genetically altered Omi / HtrA2 allele. These Hybrids have one opposite to the dHPLC column Homoduplex a changed one Retention behavior that with a probability of over 95% can be detected.

The Analysis time for a fragment is about four to five minutes, so the dHPLC a very inexpensive and time-effective screening method, for example, before an additional Sequencing of the amplificates, represents. Information about this Methods are found, for example, in McCallum, C.M. et al., (2000), Targeted screening for induced mutations. Nature Biotechnology, 18, 455-457.

Alternatively, the genetic modification may be screened by means of the fast direct sequencing method referred to as pyrosequencing (see: www.pyrosequencing.com; the content of the homepage is by reference herein). Also can be invented according to the established restriction fragment length polymorphism (RFLP) analysis apply. Also suitable is single-strand conformation (SSCP) analysis, which, however, is less sensitive and more time-consuming in terms of detection level. It is also conceivable to carry out the direct sequencing. This method is highly sensitive, but relatively costly and time consuming.

at the method according to the invention it is preferable if the investigation is based on the presence of the Nucleic acid molecule in step (b) by hybridization technology, preferably as a hybridization probe, a nucleic acid molecule is used, which is a having the sequences selected from the group consisting from SEQ ID NO: 18 to SEQ ID NO: 21 according to the enclosed sequence listing.

To In one variant, a nucleic acid molecule is used as a hybridization probe, under stringent conditions to an above-mentioned Nucleic acid molecule binds.

The In their work, inventors were able to develop nucleic acid molecules via which when used as a hybridization probe highly specific an exchange of deoxyguanosine monophosphate (dGMP) or guanine (G) against deoxythymidine phosphate (dTMP) or thymine (T) at position 421 of the open reading frame of the Omi / HtrA2 gene can be demonstrated, namely Nucleic acid molecules that have the nucleotide sequence SEQ ID No. 18 or SEQ ID No. 19. Of course this detection is also possible with complementary nucleic acid molecules that hybridize to the corresponding antisense strand of the Omi / HtrA2 gene.

there is a nucleic acid molecule with the Sequence SEQ ID NO: 18 capable of highly specific to the sense strand of the exon 1 of the Omi / HtrA2 gene to hybridize while a Nucleic acid molecule with a Nucleotide sequence SEQ ID NO: 19 highly specific to the antisense strand of the exon 1 of the Omi / HtrA2 gene can hybridize.

As the inventors have been able to show further, a nucleic acid molecule which is suitable for the Sequence SEQ ID NO: 20 or SEQ ID NO: 21, as a hybridization probe, with the replacement of deoxyguanosine monophosphate (dGMP) or Guanine (G) against deoxyadenosine monophosphate (dAMP) or adenine (A) at position 1195 of the open reading frame of the Omi / HtrA2 gene can be. The same applies to correspondingly complementary Nucleic acid molecules.

The Nucleic acid molecule with the Nucleotide sequence SEQ ID NO: 20 binds highly specifically to the sense strand of exon 7, the nucleic acid molecule with the Nucleotide sequence SEQ ID NO: 21 highly specific to the antisense strand of exon 7 of the Omi / HtrA2 gene.

by virtue of the particular suitability, for example as a PCR primer or hybridization probe, The subject of the present invention is also a nucleic acid molecule which has a Having nucleotide sequence selected from the group consisting from: SEQ ID No. 1 to SEQ ID No. 21 according to the enclosed sequence listing.

object The present invention further provides a kit comprising at least one having the above listed nucleic acid molecules.

In such a kit can in addition to the nucleic acid molecules of the invention, i. PCR primers or hybridization probes, all for the implementation of the inventive method required reagents, chemicals and buffer substances as well a detailed Description of the to be performed according to the invention or be included in another method. This has the particular advantage that thereby a flawless working, in particular of large laboratories ensured with trained personnel, i. handling errors For example, when preparing the buffer, in the implementation of the method, etc., be largely avoided.

The present invention further provides a method for finding substances that are genetically modified against the wild-type human Omi / HtrA2 protein, comprising the following steps: (a) contacting a peptide that is derived from the genetically modified Omi / HtrA2 protein, with a test substance under conditions permitting binding of the test substance to the peptide; and (b) determining whether binding of the test substance to the peptide has occurred, the genetic alteration being amino acid replacement at amino acid position 141 and / or at amino acid position 399 of the omi / HtrA2 protein is, by an alanine molecule against a serine molecule or a glycine molecule against Serine molecule is replaced.

The newly discovered correlation of amino acid position 141 and 399, respectively, genetically modified Omi / HtrA2 protein with the clinical appearance of Parkinson's disease does that so changed Protein becomes a potential target for a targeted pharmacological Intervention by means of selectively targeted substances. The above described new method now for the first time creates the possibility find such substances that target the altered Omi / HtrA2 protein and therefore have high therapeutic potential and Largely avoid side effects.

there the inventors have realized that for finding such substances the provision of such a peptide is sufficient, which is different from the genetically modified Omi / HtrA2 protein derives, but still the genetically modified amino acid position 141 and 399, respectively. Thereby, the above-described inventive method additionally simplified.

The substance to be tested may be in any conceivable chemical, biochemical or biological form, i. as a molecule, like one chemically defined compound, a peptide, protein, antibody, aptamer or as an ion or atom.

To This new procedure will determine if the test substance is on the peptide binds, i. whether a state prevails in which the testing substance at least in the immediate vicinity to the peptide and therefore possibly the activity of this Peptides can affect.

step (B) is carried out by means established in the art molecular biological and biochemical methods, for example affinity chromatographic or electrophoretic Techniques.

Conditions, which allow the binding of the test substance to the peptide, are well known in the field of protein or enzyme biochemistry; These conditions are, for example, through the use of conventional created physiological or biological buffer systems, such as For example, Tris, Hepes, or PBS-based Buffering, for example. With the addition of various salts in suitable Concentrations, as well as other usual Agents.

On Reason of the extremely significant pharmacological properties of such a substance found for one Treatment of Parkinson's disease is the subject of this present Invention also means of this method described above substance found, as well as a substance containing this substance, preferably pharmaceutical composition containing a pharmaceutically acceptable Carrier as well optionally contains further auxiliaries. Suitable pharmaceutical carriers and adjuvants are known in the art; see. Kibbe, A.H., (2000), "Handbook of Pharmaceutical Excipients ", 3rd edition, American Pharmaceutical Association and Pharmaceutical Press.

Further Advantages and characteristics of the invention will become apparent from the following embodiments and the figures.

It it is understood that the above and the following yet to be explained Features not only in the specified combination, but also usable in other combinations or alone are without departing from the scope of the present invention.

The Invention will now be described with reference to exemplary embodiments and illustrations explains in which

1 shows the exon 1 mutation found by dHPLC (G421T → A141S);

2 shows the confirmation of the exon 1 mutation by direct sequencing;

3 the. Confirmation of the exon 1 mutation by means of. shows direct pyrosequencing;

4 shows the exon 7 mutation found by dHPLC (G1195A → G399S);

5 shows the confirmation of the exon 7 mutation by direct sequencing;

6 shows the confirmation of the exon 7 mutation by RFLP analysis;

7 shows the result of the cytotoxicity test using staurosporine, and

8th shows the result of the cytotoxicity test using MG132.

Example 1: Mutation Analysis of the Omi / HtrA2 gene

411 and 514 patients suffering from Parkinson's disease and over 300 healthy controls were sampled and isolated from these according to standard procedures. The DNA was amplified by PCR. The PCR primers used here are listed in Table I below: TABLE I

With the amplificates were screened for mutation. The Mutation screening was performed using the dHPLC mutation detection systems carried out by Transgenomic (WAVE). The dHPLC uses the different Melting behavior of homo- and Heteroduplex DNA. Here, double-stranded DNA is purified by TEAA (triethylammonium acetate) to an HPLC column bound and by an increasing acetonitrile gradient of the column replaced. The DNA concentration of the acetonitrile buffer is determined by the column of detected by a laser. Come in a double-stranded DNA strand heterozygous base changes on one strand at opposite Bases, it comes through the instability at this point to a premature detachment of the HPLC column, resulting in a shift of the detection peak on the WAVE system effect. Due to differences in the separation time of the DNA, the Mutations are detected.

The used DNA is as a PCR amplificate of patients or healthy Control individuals, each exon being amplified individually. To possible To detect mutations of an exon as heteroduplex becomes PCR product denatured before use on the WAVE device and at slower rates Abkühling renatured again. It can Wild-type and mutated DNA strands adhere to each other store and train heteroduplices. Because homozygous mutations was not detected, taking into account the relative Rarity of parkinsonian mutations DNAs from two patients pooled together and then using measured by dHPLC. As a result, conspicuous detection peaks always The results of two patient samples are the respective ones Patient samples again with reference DNAs (obtained from the Center d'études des polymorphismes humaines "(CEPH), Paris, France). The here still conspicuous samples were directly sequenced on a Beckman capillary sequencer.

• * Puffer B = 0,1 M Triethylammoniumacetat (TEAA), 25% Acetonitril

The optimized dHPLC conditions for the developed PCR primer pairs are summarized in Table II below Table II

• * Buffer B = 0.1 M triethylammonium acetate (TEAA), 25% acetonitrile

Using this method, two mutations in the Omi / HtrA2 gene could be identified. The first mutation is a nucleotide exchange mutation in exon 1 of the gene in which at position 421 of the open reading frame a dGMP has been replaced by a dTMP (cf. 1 ), which at the protein level at position 141 results in an exchange of an alanine for a serine. This mutation was found in 25 of a total of 414 Parkinson's disease patients and exclusively in a heterozygous state.

Furthermore, a second mutation could be found in the Omi / HtrA2 gene, in which a dGMP was exchanged for a dAMP at position 1195 of the open reading frame in exon 7 (cf. 4 ), which results at the protein level in an exchange of glycine for serine at position 399. This mutation was found in 4 out of a total of 514 Parkinson's disease patients and also only in a heterozygous state.

The presence of the mutation in exon 1 was verified by direct sequencing (cf. 2 ). Furthermore, the presence of this mutation could be confirmed by pyrosequencing (cf. 3 ); Here, biotinylated variants of the PCR primers listed in Table I were used.

The nucleotide exchange mutation in exon 7 was also determined by direct sequencing (cf. 5 ) and further confirmed by restriction fragment length polymorphism (RFLP) analysis after MvaI digestion. In the wild-type sequence, following restriction of the amplicon of exon 7 (163 base pairs) with MvaI, three fragments are formed: 113 bp, 44 bp and 6 bp. By the base exchange of G to A in position 1195 of the coding sequence, a previously existing restriction cleavage site for the enzyme is lost (cf. 6 ).

Around the relevance of the two mutations found in terms of To determine Parkinson's disease, chromosomes were of age-correlated healthy Control subjects to both mutations by means of the aforementioned Procedure screened. In this procedure could be healthy in 740 chromosomes No exon 7 mutation can be detected in controls. In 662 chromosomes of healthy controls, only in 10 cases the exon 1 mutation can be found (p = 0.05).

The Presence of one of the two mutations found correlates accordingly with a disease or predisposition to Parkinson's disease. Both mutations are therefore valuable diagnostic markers for Parkinson's disease.

Example 2: Cytotoxicity tests with cells that are genetically modified Express Omi / HtrA2 protein

Around the pathogenetic relevance of the two found in the Omi / HtrA2 gene Analyzing mutations was by means of a cytotoxicity test Examines whether cells have the corresponding genetically modified Omi / HtrA2 protein express, more sensitive to Cytotoxins are, i. with appropriate exposure increased in to enter apoptosis.

This Test was carried out using the company's Cytotoxicity Detection Kit (LDH) Roche performed. As a measure of apoptosis In this case, after contact with the toxins, it is used by the apoptotic ones Cells released from the cytosol into the surrounding medium lactate dehydrogenase (LDH). This in turn will be over the reaction of a tetrazolium salt to formazan, the changing color from yellow (tetrazolium salt) to red (Formazan) using an ELISA reader at a wavelength of 490 nm. As a reference balance, the absorbance at 650 nm is measured.

in the 70,000 HEK293 cells (cells, the wild-type Omi / HtrA2 protein or mutant Omi / HtrA2 protein stably express; prepared according to standard methods, e.g. described in Sambrook and Russell (2001), Molecular cloning - a laboratory manual, Cold Spring Harbor Laboratory Press, New York, its content by Reference is included in the description) seeded in a 24-well plate. The cells were for 24 hours under normal culture conditions in DMEM (Invitrogen), 10% inactivated fetal calf serum (Invitrogen), 1% penicillin / streptomycin (Invitrogen). Subsequently the culture medium is taken off and against LDH assay medium (DMEM), 1% inactivated fetal Calf serum, Penicillin / streptomycin substitutes which as cytotoxin either 0.5 μM staurosporine or 3.0 μM MG132 or as a negative control solvent. The test was there as a triple test (each for poisoned cell lines and Controls). As background value for the ELISA reader, the appropriate medium was transferred to an empty well and incubated with the cells.

at Using MG132, a 24-hour incubation was performed when used of staurosporine was incubated for 6 hours. After these Incubation times were 150 μl per well removed and centrifuged. The centrifugation step served detached for sedimentation Cells by later Apoptosis could have altered the real LDH concentration. After centrifugation 100 μl of the supernatant transferred to a 96-well plate. The remaining 50 μl were resuspended and placed in the appropriate wells of the 24-well plate returned.

Around to detect the total amount of LDH, which is in relation to the Total number of cells per well, were then per well 50 ul of 10% Triton X-100 solution pipetted to lyse the still living cells. After a Incubation time of 20 minutes were then again 100 ul of centrifuged supernatant pipetted into the 96-well plate. In every well to be measured 96-well plate was then 100 .mu.l LDH assay medium and incubated at room temperature for 20 minutes.

To After this incubation period, the reaction was stopped by adding 50 μl of 1N HCl (Sigma) stopped and measured by the ELISA reader. Of the LDH value then results from the quotient of the absorption of the first and second supernatant of a well and can therefore be used as a value for the share Cells are used. The mean of three equally treated Wells gives the fraction of dead cells for standard deviation measurement.

The result of such an experiment is in the 7 and 8th shown. It was found that the HEK293 cells expressing the exon 7, ie the G1195A mutation (G399S), were more than five times more sensitive to staurosporine than the wild-type HEK293 cells; 7 , see. first pillar with fifth pillar from the left.

Furthermore, the HEK293 cells expressing the exon 1, ie G421T mutation (A141S), were found to be approximately 1.4 times more sensitive to MG132 than wild-type HEK293 cells; 8th , see. first pillar with third pillar from the left.

Thus, the two newly discovered mutations in the human Omi / HtrA2 gene are genetically important for the integrity of the cells. The incubation of such genetically modified cells with cytotoxins leads to an increased occurrence of these in apoptosis. The inventors thus discovered for the first time such genetic mutations associated with Parkinson's disease, which are both in the disturbed protein degradation - the Omi / HtrA2 protein Be Part of pathognomonic protein aggregates, so-called Lewy bodies, which the inventors were able to confirm by their own experiments - and, as the data discussed above, are involved in the regulation of apoptosis.

By providing the inventive teaching therefore not only a diagnostic tool for finding a disease or predisposition for Parkinson's disease is provided, but also created the basis for the development of a model system, by means of which, for example, found new therapeutically effective Antiparkinsonsubstanzen or the molecular pathology basics Morbus Parkinson's disease can be better understood. SEQUENCE LISTING

Claims (21)

Nucleic acid molecule coding for a human Omi / HtrA2 protein, the at amino acid position 141 and / or 399 one opposite the wild-type genetic change has, as well as for corresponding sections thereof, for use in diagnosis Parkinson's disease and / or predisposition thereto and / or for finding and testing substances that are active against Parkinson's disease. Nucleic acid molecule according to claim 1, characterized in that the genetic modification an amino acid exchange is. Nucleic acid molecule according to claim 2, characterized in that by the amino acid exchange at position 141 an alanine molecule against a serine molecule and / or by the amino acid exchange Position 399 a glycine molecule against a serine molecule is exchanged. Use of the nucleic acid molecule according to one of claims 1 to 3 for the diagnosis of Parkinson's disease and / or a predisposition thereto. A host, namely a transgenic non-human mammal, preferably a transgenic mouse, a transgenic rat, a transgenic sheep, a transgenic goat or a transgenic cow, into which the nucleic acid Molecule according to one of claims 1 to 3 was introduced. (Poly) peptide encodes the nucleic acid molecule after one the claims 1 to 3. Method for the diagnosis of Parkinson's disease in a human beings, comprising the following steps: (A) Providing a biological sample of the animal; (B) Examination of the biological sample for the presence of a Nucleic acid molecule and / or a (poly) peptide, and (c) correlation of a positive finding with a disease of Parkinson's disease and / or with a predisposition for one Disease of Parkinson's disease, characterized in that in step (b) the nucleic acid molecule of the nucleic acid molecule according to one of claims 1 to 5, and / or the (poly) peptide the (poly) peptide according to claim 8 corresponds. Method according to claim 7, characterized in that that the investigation on the presence of the nucleic acid molecule in step (b) by PCR technology. Method according to claim 8, characterized in that in that a nucleic acid molecule is used as PCR primer which has one of the sequences selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 17 according to the enclosed Sequence Listing. Method according to claim 9, characterized in that a nucleic acid molecule is used as PCR primer which binds under stringent conditions to a nucleic acid molecule, having one of the sequences selected from the group consisting from: SEQ ID No. 1 to SEQ ID No. 17 according to the attached sequence listing. Method according to one of claims 8 to 10, characterized that the PCR amplificates by denaturing high-pressure liquid chromatography (dHPLC), heteroduplex or direct sequencing become. Nucleic acid molecule containing a having the sequences selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 17 according to attached Sequence Listing. Use of the nucleic acid molecule according to claim 12 for amplification of the human Omi / HtrA2 gene. Method according to one of claims 7 to 11, characterized that the investigation on the presence of the nucleic acid molecule in step (b) using hybridization technology. Method according to claim 14, characterized in that that a nucleic acid molecule is used as the hybridization probe, which is a having the sequences selected from the group consisting of: SEQ ID NO: 18 to SEQ ID NO: 21 according to attached Sequence Listing. Method according to claim 14, characterized in that that a nucleic acid molecule is used as the hybridization probe, the binds stringent conditions to a nucleic acid molecule which is one of the sequences which has selected is selected from the group consisting of: SEQ ID No. 18 to SEQ ID No. 21 according to enclosed Sequence Listing. Nucleic acid molecule containing a having the sequences selected from the group consisting of: SEQ ID NO: 18 to SEQ ID NO: 21 according to attached Sequence Listing. Use of the nucleic acid molecule of claim 17 for detection a nucleic acid molecule encoding for humane Omi / HtrA2 protein as well as for corresponding sections thereof. Use of the nucleic acid molecule of claim 17 for detection of the nucleic acid molecule one of the claims 1 to 3. Kit, the at least one of the nucleic acid molecules after one of the claims 12 or 17 has. Method for finding genetically modified human Omi / HtrA2 protein binding substances, comprising the steps of: (a) contacting a peptide derived from the genetically altered Omi / HtrA2 protein with a test substance under conditions that allow binding of the test substance to the peptide, and (b) determining whether a binding of the test substance to the peptide has taken place, characterized in that the genetic modification is an amino acid replacement at amino acid position 141 and / or at amino acid position 399 of the Omi / HtrA2 protein, through which an alanine molecule against a serine molecule or a glycine molecule has been replaced by a serine molecule.